1. Field of Invention
The invention generally relates to methods and systems for multiplex DNA analysis in an integrated biochip, and, more specifically, to methods and systems that enable multiple target DNA fragments in a sample to be detected while reducing interference between the detected targets.
2. Description of Related Art
Integrated biochips, also referred to as microfluidics or lab-on-a-chip, have gained increasing attention in recent years especially in clinical diagnostics since they are amiable for self-contained, portable, point of care devices. These devices enable performing several biotechnology tasks in a single device thereby minimizing the need for a large laboratory setup and skilled laboratory personal to perform diagnostic and research tests. Some of the popular applications of biochips include cancer detection, environmental testing, forensics, pathogen identification, gene-expression, SNP detection, to name a few.
To date there have been several demonstration of integrated biochips, some of which illustrate processing sample for cell lysis, DNA extraction, PCR (polymerase chain reaction) or isothermal amplification and detection. We use the genetic term “amplification” to mean PCR and isothermal amplifications. These terms are interchangeable unless specified otherwise. For example, the integration of sample preparation and amplification to detection strategies such as capillary electrophoresis, mass spectroscopy, qPCR, and microarray have been widely demonstrated. However, it is known that the final DNA fragment detection method determines the limitations on the number and size of the DNA fragments that can be amplified by PCR or isothermal amplification.
Typically, one amplified DNA fragment can correspond to one organism/disease identification. Hence, the ability to amplify several DNA fragments and uniquely detect each of these fragments provides a better platform for identification from a single experiment, both for research and clinical diagnostics. In this regard, in recent years multiplex PCR and isothermal amplification reactions have gained attention since it is capable of generating several DNA fragments to identify many organisms/diseases from a single reaction. Nevertheless, it is the DNA detection strategy employed post multiplex amplification that determines the number (or size) of the DNA fragments that can be uniquely detected.
Capillary electrophoresis (CE) and mass spectroscopy are popular approaches for detecting several DNA fragments generated by multiplex PCR/isothermal amplification. However, both these methods impose the limitation that the amplified DNA fragments must be of unique sizes and the sizes of the DNA fragments should be larger than the separation resolution of the instrument/system. Furthermore, both capillary electrophoresis and mass spectroscopy suffer from an inability to discriminate non-specific amplification if those non-specific DNA fragments are similar in size to any of the other DNA fragments generated by multiplex PCR/isothermal amplification. Also, since capillary or microfluidic electrophoresis in biochips utilize micro fluidic path for DNA migration, they require expensive excitation and emission optics (e.g., lasers, CCD, photomultipliers) to sensitively detect low concentration fluorescence in biochips. Nevertheless, the proven approach of CE for DNA fragment analysis has rendered this method for the widespread use in integrated biochips for over a decade.
In contrast, traditional qPCR using the TaqMan probe method has better specificity but is limited to the detection of DNA fragments that can be labeled by uniquely colored fluorophore, determined by the instrument color detection capability (e.g., known systems are believed to be limited to nine colors). Hence, integrated biochips that utilize the qPCR method are limited to scanning and identifying fewer than nine DNA fragments, if the capability of a nine color qPCR system is employed. However, qPCR systems typically only demand low cost excitation and emission optics (e.g. LED, flash lamps, and photodiodes) and this enables low-cost instrumentation for this method.
Another approach that increases detection capability for multiplex PCR/isothermal amplification is the use of microarray technology coupled with PCR/isothermal amplification. In such a method, a biochip that performs PCR or isothermal amplification is then coupled with a microarray method downstream. The feasibility to spot several hundred detection regions in the microarray technology makes it appealing for multiplex PCR/isothermal amplification. However, similar to most high sensitivity electrophoresis systems, microarray detection also requires the use of a high power spot-excitation source for the fluorophores hence demanding the use of expensive fluorescence excitation and optical detection components (e.g. lasers, CCD, photomultipliers).
From an instrument cost perspective, of the numerous commercially available instruments, qPCR systems (e.g. Applied Biosystems, BioRad, Roche, Eppendorf) are typically several-fold less expensive than electrophoresis (e.g. ABI, Qiagen, Agilent) and microarray systems (e.g. Affymetrix, Molecular Devices, Roche, Tecan) for comparable applications.
Meanwhile, the DNA fragment detection strategy employed in the integrated biochip, methods, and systems described herein enable a much higher degree of multiplexing as compared to traditional qPCR, while only requiring similar inexpensive optics conventionally used in qPCR. The increased fragment detection capability enables the disclosed techniques to identify more organisms/diseases from a single test as compared to the popular and conventional qPCR technology. Thus, embodiments of the invention enable the use of the relatively low-cost instrumentation of qPCR optics while increasing the fragment detection capability by several folds.
Furthermore, certain embodiments of the invention include universal fit Luer taper-type connectors integrated into the surface of the biochip for quick connection while maintaining an air/water-tight seal of reagent pre-filled syringes/cartridges to the biochip.